OBJECTIVE: The objective of this research was to investigate
neurodevelopmental mechanisms underlying previously observed behavioral
impairments observed after neonatal exposure to polybrominated diphenyl
ethers (PBDEs).

Fetal and neonatal exposure to neurotoxicants have adverse effects
on neurodevelopment. Early (small) effects of xenobiotics on the brain
could aggravate these effects during development, creating a critical
window for neurotoxicity. However, the underlying mechanisms are not
well understood (Szpir 2006). Recently, a range of behavioral and
neurochemical effects have been described for polychlorinated biphenyls
(PCBs) (for review, see Fonnum et al. 2006; Mariussen and Fonnum 2006).
Nowadays, the increasing concentrations of the structurally related
polybrominated diphenyl ethers (PBDEs) in the environment, human food
chain, and human tissues (Hites 2004) raise concern about possible
neurotoxic effects. In most samples,
2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is the predominant
congener. PBDEs are used as flame retardants in a range of products,
including electronic equipment, furniture, construction materials, and
textiles.

Of concern is that children, at the age of early brain development,
accumulate BDE-47 more rapidly than adults because of their diet
(breast-feeding/relatively large intake) and behavior (contact with
house-dust) (Jones-Otazu et al. 2005). Distribution studies show that
developing mice reach higher tissue concentrations of BDE-47 compared
with adult mice after identical dosing regimens (Staskal et al. 2006).
Behavioral studies have demonstrated adverse neurodevelopmental effects
on learning and memory after neonatal BDE-47 exposure. Habituation capability in mice, studied by scoring spontaneous behavior after
placement in a new environment, is reduced and this effect is
long-lasting and increases with age (Eriksson et al. 2001).

Recently, a proteomics approach was used to investigate the effect
of a single oral dose of 12 mg (21.2 [micro]mol)/kg body weight (bw)
2,2',4,4',5-pentabromodiphenyl ether (BDE-99) on brain protein
levels in mice, 24 hr after exposure. Levels of striatal proteins
associated with neurodegeneration and neuroplasticity and of hippocampal
proteins associated with metabolism and energy production were found to
be changed (Alm et al. 2006). It is unclear whether such changes occur
after exposure to other congeners, and whether these protein changes
have functional consequences.

The main objective of our study was to gain insight in the
mechanisms underlying the observed effects of BDE-47 on learning and
memory (Eriksson et al. 2001). To this purpose we have investigated
N-methyl-D-aspartate (NMDA)-dependent long-term potentiation (LTP) in
hippocampal slices from animals exposed to a dose of BDE-47 known to
induce behavioral aberrations. NMDA-dependent LTP has been used as an
electrophysiologic substrate for learning and memories for many years.
This form of LTP is induced by tetanic stimulation, strong
depolarization, and a large increase in intracellular [Ca.sup.2+] level
(for review, see Lynch 2004; Malenka and Nicoll 1999; Soderling and
Derkach 2000). Paired pulse facilitation (PPF), a form of short-lasting
plasticity that presumably reflects presynaptic function (Xu-Friedman
and Regehr 2004), was investigated to reveal possible presynaptic
effects of BDE-47. In additional ex vivo experiments, we investigated
protein expression levels in the postsynaptic density (PSD) and
catecholamine release from chromaffin cells to further reveal underlying
mechanisms. Acute effects of BDE-47 on intracellular [Ca.sup.2+] and
catecholamine release of PC12 cells have been studied in vitro to assess
the involvement of transient acute effects on potential presynaptic
targets. Our findings provide a functional basis for previously observed
neurobehavioral changes (Eriksson et al. 2001).

Materials and Methods

Animals and chemicals. Male C57Bl/6 mice pups (litters culled to 5
pups each) with mother (Harlan, Horst, the Netherlands) were housed in a
standard animal facility on a 12-hr light/dark cycle with food and water
ad libitum. Animals were treated humanely and with regard for
alleviation of suffering. All experimental procedures were performed
according to Dutch law and approved by the Ethical Committee for Animal
Experimentation of Utrecht University.

BDE-47 was synthesized and purified (~ 99%) at the Wallenberg
laboratory of Stockholm University. For oral dosing, BDE-47 was
dissolved in the egg lecithin/peanut oil mixture and sonicated with
water to obtain a 20% (wt/wt) fat:water emulsion.

Extracellular recording of field potentials. We recorded f-EPSPs in
the CA1 region of hippocampal slices as previously described by Van der
Heide et al. (2005), with minor modifications. Slices were superfused
with carbogenated ACSF (~2 mL/min) in a recording chamber at
30[degrees]C. A bipolar stainless steel stimulation electrode ([empty
set] 0.1 mm) was placed on the afferent fibers of the stratum radiatum
of the hippocampal CA1 region, as shown in a Nisslstained hippocampal
slice in Figure 1A. f-EPSPs were recorded with ACSF-filled glass
microelectrodes using an Axoclamp-2B amplifier (Axon Instruments, Foster
City, CA, USA). Data were digitized and stored using "Spike2"
software (Cambridge Electronic Design, Cambridge, UK).

Stimulation intensities for threshold and maximum f-EPSPs were
determined. Slices with a maximum response amplitude of [greater than or
equal to] 1 mV were included in the experiment. During baseline
recording, half-maximum f-EPSPs were evoked every 30 sec. After 15 min
baseline recording, LTP was induced with a single tetanic stimulation
(100 Hz, 1 sec) and f-EPSPs were recorded for another 30 min. PPF, with
interstimulus intervals of 50, 100, 200, 500, and 1,000 msec, was
recorded under identical conditions as for LTP.

For data analysis, we determined initial slopes of the f-EPSPs
(Figure 1B). For quantification of LTP, the slope was normalized against
the average f-EPSP slope during baseline. Average relative increase of
the slope was determined 20-30 min after tetanic stimulation as a
measure for LTP and 0-7.5 min after tetanic stimulation as a measure for
posttetanic potentiation (PTP) in the individual animals. To determine
PPF, paired-pulse ratio (PPR) was determined by dividing the slope of
the second average f-EPSP by the slope of the first average f-EPSP (n =
10).

Statistical analysis. All data are presented as mean [+ or -] SE.
PC12 data were compared using Student's paired t-test. We first
compared the LTP data using a two-way analysis of variance (ANOVA) with
post hoc Bonferroni testing (Sigmastat software; Systat Software Inc,
Erkrath, Germany), followed by additional unpaired t-tests to specify
the effects on PTP and LTP. We used unpaired Students' t-test for
all other data.

Results

Pups exposed to BDE-47 did not differ in body weight and relative
thymus weight compared with their unexposed littermates (data not
shown), indicating the absence of general toxicity, treatment-dependent
food competition, extensive immune suppression, and stress.
Additionally, visual inspection of the brain slices of exposed pups did
not show any changes of general hippocampus morphology (data not shown).

Figure 2 shows the results from f-EPSP recordings in the CA1 region
of mouse hippocampus for control and BDE-47-exposed groups. No
differences in stimulus-response relation were seen. No effects were
observed on half-maximum f-EPSP slopes before LTP induction (control:
682 [+ or -] 138 V/sec; BDE-47 exposed animals: 679 [+ or -] 92 V/sec).

After tetanic stimulation, an immediate large increase of the
f-EPSP is apparent, although the increase is significantly lower in the
BDE-47-exposed group than in the control group. The increase of the
f-EPSP during the first 7.5 min post-tetanus is classified as PTP. In
the BDE-47-exposed mice, there was significantly less PTP (135 [+ or -]
9%) than in the control mice (190 [+ or -] 17%) (p < 0.01) (Figure
2). After PTP the f-EPSP size decreases but stabilizes at a higher level
than baseline. This level of LTP is maintained for at least 30 min. In
the BDE-47-exposed mice, LTP was significantly lower (130 [+ or -] 7%)
than in the control group (165 [+ or -] 16%) (p < 0.05). The
significance of these findings was confirmed by two-way ANOVA with post
hoc Bonferroni testing. The trace inset illustrates the enhancement of
f-EPSPs after tetanic stimulation. The cumulative probability curve of
LTP in the individual experiments (Figure 2) indicates a shift to lower
LTP values in the BDE-47 group.

Figure 3 shows the effect of BDE-47 on PPF at different
interstimulus intervals. For the 50-msec interstimulus interval, the PPR
was 1.98 [+ or -] 0.11% in the control group and 1.87 [+ or -] 0.15% in
the BDE-47 group. For the 1,000-msec interstimulus interval, the PPR was
decreased to 1.16 [+ or -] 0.03% in the control group and 1.08 [+ or -]
0.03% in the BDE-47 group. Insets show representative recordings of PPF.
No effects of BDE-47 on PPR were detected.

Because activation of NMDA receptors is required for LTP, the
reduction of LTP in BDE-47-treated mice could reflect an alteration of
NMDA receptor-associated signaling elements. Because the NMDA receptor
complex is enriched in the PSD, we used Western blot analysis to measure
protein levels of NMDA receptor subunits and other PSD-associated
signaling proteins in total homogenate and TIF, representing the PSD
compartment by Western blot analysis (Gardoni et al. 2001). Protein
composition of this preparation was carefully tested for the absence of
presynaptic markers and enrichment in PSD proteins (Figure 4A; Gardoni
et al. 2001). Representative Western blots for all investigated proteins
in hippocampal homogenate and TIF are also shown in Figure 4B. BDE-47
had no effects on protein levels in cortical homogenate and TIF (data
not shown) and hippocampal homogenate (Figure 4C). Figure 4C shows
amounts of the proteins in TIF of the hippocampus of BDE-47-exposed mice
compared with control mice. Significant changes in protein levels of
NMDA receptor subunits NR1 and NR2A, and NMDA receptor interacting
proteins PSD-95 and SAP97 were not detected. However, protein levels of
NMDA receptor subunit NR2B (75 [+ or -] 2%) and
[alpha]-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA)
receptor subunit GluR1 (71 [+ or -] 4%) were significantly reduced (p
< 0.01). There was a significant decrease in the
autophosphorylated-active form of [alpha]CaMKII (p286-[alpha]CaMKII) to
65 [+ or -] 8% of control level (p < 0.05), although total
[alpha]CaMKII was not changed.

In the experiments described above, postsynaptic effects of BDE-47
are observed, whereas presynaptic functional effects are not detected.
However, possible effects on presynaptic mechanisms might remain
undetected at a dose of 6.8 mg (14 [micro]mol)/kg bw BDE-47. To
ascertain the apparent absence of presynaptic effects of BDE-47, we
investigated effects on catecholamine release in chromaffin cells
obtained from mice exposed to vehicle or to a higher dose (68 mg (140
[micro]mol)/kg bw) of BDE-47. No changes were detected in the different
parameters of vesicular catecholamine release; that is, basal and
high-[K.sup.+] evoked release frequency and vesicular release parameters
like quantal size (vesicle content), spike amplitude, and 50-90% rise
time (data not shown).

Additional in vitro experiments were performed in PC12 cells to
investigate acute effects of BDE-47 exposure on calcium homeostasis and
release mechanisms. Figure 5A shows the average F340/F380 ratio in PC12
cells during bath application of DMSO, 2 [micro]M BDE-47, and 20
[micro]M BDE-47 normalized to baseline (first 5 min). The higher
concentration of BDE-47 induced an increase in normalized F340/F380
ratio (t = 12-24 min, p < 0.01). To investigate whether the increase
in intracellular [Ca.sup.2+] has functional consequences, vesicular
catecholamine release was also investigated (Figure 5B). The average
number of amperometrically recorded events of vesicular release amounted
to 1.9 [+ or -] 0.7 events/min (n = 9) in control experiments. During
superfusion with 20 [micro]M BDE-47, the release frequency was enhanced
to 6.0 [+ or -] 1.7 events/min (n = 6; p < 0.05), whereas superfusion
with 2 [micro]M BDE-47 caused no detectable effect (1.2 [+ or -] 0.5
events/min; n = 7). BDE-47 had no effect on release evoked by
high-[K.sup.+] depolarization of the cells. Differences in vesicular
release parameters could not be detected (data not shown).

Discussion

A broad spectrum of neurotoxicants (e.g., environmental pollutants
such as metals, pesticides, and PCBs) has been shown to cause a
reduction of habituation after neonatal exposure (Eriksson et al. 1990,
1991; Eriksson and Fredriksson 1991; Fredriksson et al. 1992). However,
from the behavioral effects it is difficult to deduce information about
underlying mechanisms.

In the present study, we found that neonatal exposure to BDE-47
causes developmental effects consisting of a reduction of PTP and LTP,
as well as specific reductions of key postsynaptic proteins involved in
glutamate receptor signaling. Presynaptic parameters were not affected
ex vivo. In vitro experiments on PC12 cells show an increase in
intracellular [Ca.sup.2+] and spontaneous vesicular release, only at the
highest concentration BDE-47 (20 [micro]M). The combined results suggest
that presynaptic changes do not directly contribute to the observed
defect in synaptic plasticity.

The exposure to BDE-47 took place during a period of rapid brain
growth, which in mice takes place during the first 3-4 weeks of life,
reaching its peak around PND10 (Davison and Dobbing 1968). The multitude
and complexity of processes during this rapid development makes the
developing brain particularly vulnerable to the effects of xenobiotics,
like the adverse effect of BDE-47 on spontaneous behavior and
habituation (Eriksson et al. 2001). Interestingly, exposure to BDE-47
does not affect performance in the Morris water maze test (Eriksson et
al. 2001), commonly used as a learning task to detect effects in the
hippocampus. This suggests that habituation is a more sensitive
parameter for BDE-47 effects in the hippocampus.

We observed a specific reduction of key proteins in the PSD (i.e.,
GluR1, NR2B, and p286-[alpha]CaMKII). Because no changes were observed
in total hippocampus homogenate, the specific decrease in the PSD is
therefore attributed to changes in glutamate receptor subunit
trafficking or clustering in the PSD instead of a reduced protein
translation.

A study in GluR1-knockout mice showed that approximately 10% of the
normal amount of GluR1 is sufficient for LTP (Mack et al. 2001). Also, a
GluR1-independent form of LTP has been observed in juvenile
GluR1-knockout mice (Jensen et al. 2003). Therefore, major effects on
LTP as a consequence of the observed reduction of AMPA subunit GluR1 by
approximately 30% are not expected.

The observed reduction of NR2B subunits results in an increased
NR2A/NR2B ratio. The majority of NMDA receptors consist of 2 NR1 and 2
NR2A or 2 NR2B subunits. NR2A-NMDA receptors gate smaller [Ca.sup.2+]
currents, have a lower affinity for glutamate, and desensitize faster
than NR2B-NMDA receptors (Kutsuwada et al. 1992). Therefore, an
increased NR2A/NR2B ratio is likely to result in a higher threshold for
LTP induction, which could explain the reduction of PTP and LTP.

In mice exposed to BDE-47, the autophosphorylated-active form of
[alpha]CaMKII was significantly reduced. Because CamKII
autophosphorylation is essential for hippocampal NMDA-dependent LTP
(Giese et al. 1998), this specific effect may lead to reduced synaptic
plasticity resulting in behavioral impairments.

To ascertain the absence of presynaptic effects, we investigated
neurotransmitter release from chromaffin cells from BDE-47-exposed [68
mg (140 [micro]mol)/kg bw] mice. Because PPR and chromaffin neurotransmitter release remained unchanged after developmental exposure
to BDE-47, and because modest acute effects on free intracellular
[Ca.sup.2+] and spontaneous vesicular catecholamine release in PC12
cells were only detected at a concentration of 20 [micro]M BDE-47, we
propose that presynaptic changes do not contribute considerably to the
observed functional defect in synaptic plasticity. Based on tissue
distribution data for 1 mg/kg bw [.sup.14.C]-BDE-47 orally given to
C57Bl/6 mice on PND10 (Staskal et al. 2006), brain concentration at
sacrifice after exposure to 6.8 mg (14 [micro]mol)/kg bw BDE-47 is
estimated to be 0.43-0.81 [micro]M and the peak brain concentration,
reached 8 hr after exposure, is estimated to be 1.1 [micro]M. These
estimated concentrations are at least one order of magnitude lower than
the lowest effective concentration in the in vitro experiments described
here.

Pure (~ 99%) BDE-47, which has been used in only a few experiments,
has revealed formation of reactive oxygen species in human neutrophils
and increased [.sup.3.H]-phorbol ester binding in primary rat cerebellar
granule neurons, also at micromolar concentrations (Kodavanti et al.
2005; Reistad and Mariussen 2005). The effects of BDE-47 in PC12 cells
reported here occur at concentrations in the same order of magnitude.

In the 1990s, an association between delayed human neurodevelopment
and prenatal or early exposure to PCBs was reported by cohort studies.
These results were corroborated by experiments demonstrating the
developmental neurotoxicity of PCBs. The observed interaction with the
thyroid hormone system is usually considered part of the underlying
mechanism (for review, see Winneke et al. 2002). For hazard
characterization of PCBs and the structurally related PBDEs, it is
relevant to investigate whether they induce similar effects through
similar mechanisms. This is of particular importance because, in
neonatal mice, the effects of a combined dose of PCB-52 and BDE-99 on
spontaneous motor behavior and habituation capability appear to be
additive or perhaps even synergistic (Eriksson et al. 2006).

High human serum concentrations of BDE-47 were measured in female
inhabitants of California by Petreas et al. (2003); the concentration of
BDE-47 in serum ranged from 5 to 510 ng/g lipid weight, with a median of
16.5 ng/g lipid weight. High concentrations (> 100 ng/g lipid weight)
have also been reported in Californian children (Fisher et al. 2006).
The highest and median values correspond (using average physiologic
values) to blood concentrations of approximately 11.5 nM and
approximately 0.37 nM. Using the tissue distribution data for 1 mg/kg bw
14C-BDE-47 (Staskal et al. 2006), the dose used in the current study
corresponds to an estimated blood concentration of approximately 2.6
[micro] after 3 hr and to approximately 0.6 [micro]M after 10 days
(i.e., ~ 50-200 times higher than in the worst, and ~ 1,600-7,000 times
higher than in the median human situation described above). For risk
assessment, the difference between the animal dose level causing an
adverse effect and the highest human dose levels is relatively small,
considering safety factors for species extrapolation and intraspecies variability. Additional uncertainty comes from the fact that humans are
exposed to multiple flame retardants over a lifetime. Accumulation of
BDE-47, as demonstrated in primary rat cerebellar granule neurons and
primary rat neocortical cells (Kodavanti et al. 2005; Mundy et al.
2004), is another reason for concern about the neurotoxic potential of
PBDEs.

No tolerable daily intake is assigned to PBDEs because sufficient
data are not available. However, the limited toxicity data suggest that
adverse effects induced by exposure to the more toxic congeners in
rodents occur at doses of at least 100 [micro]g/kg bw per day [Joint
FAO/WHO Expert Committee on Food Additives (JECFA) 2005]. The
combination of quantitative molecular data with functional
neurophysiologic effects reported here provides strong functional
support for the previously reported neurobehavioral effects (Eriksson et
al. 2001) and is essential for characterization of the neurotoxic hazard
of brominated flame retardants, particularly for rational risk
assessment, which is required in response to the general concern about
the vulnerability of the developing brain.